Materials and methods for detection and treatment of immune system dysfunctions

ABSTRACT

The subject invention concerns novel materials and methods for the treatment and/or prevention of autoimmune disease. In a specific embodiment, elevated production of prostaglandin synthase-2 (PGS-2) is correlated with autoimmune dysfunction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 08/916,586, filedAug. 22, 1997; which is a continuation-in-part of application Ser. No.08/701,928, filed Aug. 23, 1996 and now U.S. Pat. No. 5,939,069.

BACKGROUND OF THE INVENTION

Diabetes is a term that refers to a collection of diseases resulting indisordered energy metabolism and varying degrees of blood glucoseelevations or hyperglycemia. One of the best characterized forms of thedisease is that which arises from an immunologically mediateddestruction of the insulin secreting pancreatic beta cells. This severeform of the disease is termed Insulin-dependent Diabetes (IDD or IMD)since it is associated with progressive insulin deficiency andcoincident symptoms such as weight loss, glycosuria and polyuria, andincreased thirst or polydipsia. Other terms for this form of diabetesare Type 1 Diabetes (cf. Type 2 Diabetes which results from an inherentresistance to insulin action); Ketosis Prone Diabetes because there isabnormal generation of ketone bodies as a result of excessive breakdownof body fats due to the severe insulin deficiency; or Juvenile Diabetes,since virtually all diabetes that appears in childhood and adolescenceis of this type.

Diabetes is a major public health problem, especially in Westerncountries. The incidence rates vary greatly worldwide, from as high as40 per 100,000 persons in Finland to as low as 1-2 per 100,000 among theJapanese. The peak incidence is during the pubertal years, associatedwith the increasing bodily demands for insulin associated with musclegrowth. The prevalence rates in the United States population under age20 years is 0.25% and it approaches 0.4% over a lifetime, albeit anestimated 10-20% of patients with Non Insulin-dependent Diabetes (NIDD)or Type 2 or Maturity Onset Diabetes also have, in reality, slowlyprogressive IDD. Thus, it is estimated that there may be at least 1million Americans affected by IDD.

Diabetes results in progressive damage to the blood vessels of the body,to a degree that depends upon the severity of hyperglycemia and itsduration. The incident mortality rate for IDD has been calculated to be7-fold higher than for age matched non-diabetic controls. Whereas thedecade long Diabetes Control and Complications Trial (DCCT)—concluded in1994 by the National Institutes of Health in the United States—showedthat meticulous insulin replacement therapy would slow the appearance ofdamaged arteries, it was not able to completely prevent this damagesince blood glucose levels were difficult to keep within normal limits.Ocular complications of diabetes are the leading cause of new blindnessin persons 20-74 years of age. The risk of lower extremity amputation is15-fold higher in those with diabetes. Approximately 40% of personsundergoing renal transplantations have kidney failure because ofdiabetes, and the proportion due to diabetes continues to rise eachyear. Women with diabetes produce newborn infants with a 7% newbornmortality rate. Other complications of diabetes include increased heartdisease and stroke, loss of nerve cells or neurons enervating the limbsand intestine, impotence and infertility, cataract formation in the lensof the eyes, increased periodontal disease, and predisposition toinfectious diseases especially from bacteria and yeast. Of all patientswith diabetes, those with IDD have a disproportionate share of thesecomplications because of its severity and usual early age of onset. Inthe United States, the direct health care costs attributable to diabetesin 1994 have been estimated to exceed $120 billion. Thus it is importantthat the pathogenesis of IDD be understood and strategies be developedto prevent it as a fully expressed clinical disease.

Patients with IDD are unusually prone to other diseases that have becomerecognized as having autoimmune origins. These diseases includethyroiditis or Hashimoto's disease, Graves' disease, Addison's disease,atrophic gastritis and pernicious anemia, celiac disease, and vitiligo(Maclaren, N. K. [1985] Diabetes Care 8(suppl.):34-38). Evidence thatIDD itself has an autoimmune nature began with histological studies ofpatients; these studies indicated that the islets were infiltrated witha chronic inflammatory (lymphocytic) infiltrate termed insulitis. Thiswas supported in the early 1970s by reports of islet cell autoantibodiesreactive to antigens within the cytoplasm (ICA) (Lendrum et al. [1975]Lancet 1:880-882) or confined to the islet cell surfaces (ICSA)(Maclaren et al. [1975] Lancet 1:977-1000) as detectable by indirectimmunofluorescence. Later it was recognized that many patients alsodevelop autoantibodies to insulin (IAA) before their diagnosis (Palmeret al. [1983] Science 222:1337-1339) as well as to insulin receptors(Maron et al. [1983] Nature 303:817-818). Autoantibodies were alsoreported to an islet cell protein composition of 64,000 M. Wt. in man(Baekkeskov et al. [1982] Nature 298:167-169), in the Biobreeding (BB)rat model (Baekkeskov et al. [1984] Science 224:1348-1350), and in theNon Obese Diabetic (NOD) mouse model (Atkinson and Maclaren [1988]Diabetes 37:1587-1590). 64 kDa antigen has subsequently been reported tobe the lower molecular weight isoform of glutamic acid decarboxylase(GAD₆₅) (Baekkeskov et al. [1990] Nature 347:151-156) (Kauffman et al.[1992] J. Clin. Invest. 283-292). GAD is an enzyme that convertsglutamate into the membrane stabilizing neurotransmitter called gammaamino butyric acid or GABA. In addition to autoantibodies to GAD,peripheral blood mononuclear cells were shown to be autoreactive inpatients developing IDD (Atkinson and Maclaren et al. [1992] Lancet339:458-459; and Harrison et al. [1993] Lancet 341:1365-1369).

It has previously been demonstrated in several autoimmune diseases,including IDD, systemic lupus erythematosus(SLE), rheumatoid arthritis(RA), multiple sclerosis(MS), and autoimmune thyroid disease, thatantigen-presenting cells (APCs) such as monocytes and macrophages aredysfunctional in their ability to activate T lymphocytes (Via, C. S. etal. [1993] J. Immunol. 151:3914-3922; Serreze, D. [1993] FASEB J.7:1092-1096; Rasanen, L. et al. [1988] Clin. Exp. Immunol. 71:470-474;Hafler, D. A., et al. [1985] J. Neuroimmunol. 9:339-347). The defect(s)in APC function, however, have thus far not been defined at the cellularor molecular level.

Prostaglandins (PGs) are lipid molecules formed from a precursormolecule, arachidonic acid, through the actions of specific enzymescalled prostaglandin synthases (PGS-1 and PGS-2). PGS-1 mRNA and proteinare constitutively expressed, and this enzyme is responsible for theproduction of low levels of PGs and functions as a housekeepingmolecule. PGS-2 is an inducible enzyme expressed by macrophages andmonocytes during inflammation and following exposure to mitogens,cytokines, and bacterial cell wall products, i.e., lipopolysaccharide(LPS) (Farber, J. M. [1992] Mol. Cell. Biol. 12:1535-1545; Vane, J. R.[1994] Proc. Natl. Acad. Sci. USA 91:2046-2050; Kujubn, D. A. [1993] J.Biol. Chem. 266:12866-12872; Ristimaki, A. et al. [1994] J. Biol. Chem.269:11769-11775). PGS-2 has been shown to be expressed in the cellslining the joints of individuals with rheumatoid arthritis and maycontribute to the ongoing inflammation in the affected joint (Crofford,L. J. et al. [1994] J. Clin. Invest. 93:1095-1101).

In addition to prostanoids, monocytes produce monokines that stronglymodify inflammation and immune responses. Among the monokines that ingeneral upregulate or promote inflammation and immunity are TNFα, IL-1α,IL-1β, IL-12, and IL-6. Monokines that tend to downregulate theseresponses are IL-4, IL-10, IL-13, and IL-1 receptor antagonist (IL-1RA).

Prostaglandins are known to have an effect on the expression ofmonokines. For instance, prostaglandin E₂ (PGE₂) is known to suppressthe production of TNFα (Seldon, P. M. et al. [1995] Mol. Pharmacol.48:747-757; Strieter, R. M. et al. [1990] J. Leuk. Biol. 47:366-370).There are also reports that PGE₂ suppresses IL-1α (Endres, S. et al.[1991] Immunology 72:56-60; Zhong, W. W. [1995] Immunology 84:446-452).In contrast to its suppressive effects, PGE₂ production stimulates theproduction of IL-10 by monocytes (Strassman, G. et al. [1994] J. Exp.Med. 180:2365-2370). IL-10 in turn modulates PGE₂ production bysuppressing PGS-2 production (Mertz, P. M. et al. [1994] J. Biol. Chem.269:21322-21329). In addition, IL-10 stimulates the production ofanother potent immunoregulatory monokine,IL-1RA (Spengler, R. N. et al.[1989] J. Immunol. 142:4346-4350). PGE₂ therefore can stimulateimmunoregulation through its own action and through its actions onmonokine production.

If monocytes are chronically exposed to PGE₂ in vitro, there is a lossof response to its action. This desensitization phenomena is mediated bythe down-regulation of PGE₂ receptors (Coffey, R. G. et al. [1990] J.Leuk. Biol. 48:557-564). For example, PGE₂ in vitro normally suppressesTNFα production, but chronic exposure leads to a loss of suppression ofthis monokine by PGE₂. Removal of, or blocking, PGE₂ can reverse thedesensitization process (Howard, M. et al. [1992] J. Clin. Immunol.12:61-784).

Currently, one can screen for individuals at high risk for thedevelopment of IDD by serologic methods only, which reflect autoimmune Band T lymphocyte activity. Serologic tests identify approximately 80-85%of individuals who have existing autoimmune disease against theinsulin-producing cells of the pancreas. Of the ICA+ population,approximately 80-85% will develop IDD within the ensuing five years.Currently no test exists, immunologic, genetic, or otherwise, which canidentify individuals at risk for IDD other than these serologic tests.

Because IDD takes several years to develop in an individual,autoimmunity may be firmly established at the time that individualsdevelop ICAs. Detection of background cellular or genetic factorsnecessary for the development of autoimmune disease and expressed earlyin the disease process is of great clinical importance. Detection ofthese factors would preferably identify individuals before autoimmunityis initiated, or perhaps at earlier stages of the disease than detectedby ICA. Earlier detection would be of great clinical importance inidentifying individuals at high risk for disease where theadministration of preventative therapies that attempt to preserve theresidual insulin-secreting cells are employed.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to materials and methods for thedetection, prevention, and treatment of diabetes, other autoimmuneconditions, and conditions involving dysfunctional apoptotic processes.In a specific embodiment, the subject invention concerns theidentification of a defect in antigen-presenting cells (APCs) that isassociated with diabetes and other autoimmune disorders. One aspect ofthis defect is the elevated production of prostaglandin synthase-2(PGS-2) in cells of individuals who have developed or will developautoimmune disease.

In a specific embodiment of the subject invention, diabetes and/or otherautoimmune disease can be predicted and/or monitored by assaying forexpression of prostaglandin synthase-2 (PGS-2) by antigen-presentingcells. Thus, one aspect of the invention is the discovery that PGS-2 isa cellular marker that is strongly associated with clinical autoimmunediseases such as IDD. In a preferred embodiment, the antigen-presentingcells which are monitored for diagnostic purposes are macrophages and/ormonocytes. Expression of PGS-2 in these cells is an indicator ofautoimmune susceptibility or disease.

The expression of PGS-2 can be detected in any of a number of ways whichwould be apparent to those skilled in the art having the benefit of thisdisclosure. For example, the expression of PGS-2 can be detected by thepresence of PGS-2 messenger RNA (mRNA), presence of the PGS-2 proteinitself, or by detecting biological effects of the PGS-2 protein, i.e.,PGE₂ production.

A further aspect of the subject invention pertains to the identificationof diagnostic markers for autoimmune disease on T-cells. In oneembodiment it has been found that CD25 expression on T-cells can becorrelated with autoimmune disease such as IDD. Specifically, it hasbeen found that individuals with a susceptibility to diabetes have alower level of expression of CD25 on T-cells. Furthermore, inhibition ofPGE₂ was found to significantly increase CD25 expression in cells ofindividuals at risk for IDD. Inhibition of PGE₂ did not enhanceexpression of CD25 in individuals who are not at risk to develop IDD. Ina further embodiment, individuals at risk for IDD have been found tohave decreased levels of FAS receptor expression compared to individualswho are not at risk to develop IDD. In a preferred embodiment T-cellswhich express both FAS receptor and CD25 are examined for a reducedlevel of expression of these proteins. A reduced, level of expression ofFAS receptor and CD25 compared to normal controls is indicative ofdiabetes or other autoimmune conditions.

Assays for PGS-2 expression, CD25 expression and/or FAS receptorexpression add new dimensions to disease prediction such as assessingdisease activity and progression, and predisposition for developingother autoimmune diseases.

The diagnostic procedures described herein can be used to detectevidence of autoimmune dysfunction before the appearance of clinicalsymptoms. This early detection makes it possible to initiate appropriatepreventative measures.

A further aspect of the subject invention is the discovery thatpharmacologic inhibition of PGS-2 has potent inhibitory effects on thedevelopment of autoimmune disease. In one embodiment, prevention of IDDcan be achieved according to the subject invention by drug therapy thatblocks PGS-2 enzymatic activity. PGS-2 inhibitors provide aninexpensive, safe, and well-tolerated approach to the prevention of thisdisease. Furthermore, the efficacy of therapy can be monitored bymeasuring serum or urine PGE₂ levels. Also, for certain individuals,PGS-2-specific inhibitors were found to markedly reduce production ofPGE₂ and increase IL-1 receptor antagonist (IL-1RA) in human monocytes.

A further aspect of the subject invention pertains to treatments fordiabetes or other autoimmune disease which comprise modulating CD25expression and/or FAS receptor expression. In a preferred embodiment,individuals at risk for IDD are treated to increase CD25 expression.This increase can be effected by, for example, administering a compoundwhich inhibits PGS-2 activity, PGE₂ activity, or the activity of cyclicAMP or related compounds. Such inhibition can be achieved by, forexample, an inhibitor of PGS-1 or PGS-2. In a preferred embodiment, aPGS-2 specific inhibitor such as NS398 is administered.

A further aspect of the subject invention pertains to diagnostic andtherapeutic methods based on modulation and/or detection of cell deathmechanisms and events. In accordance with the subject invention,differences in cell death events relating to expression or activity ofPGS-2 and/or related molecules can be exploited to provide criticaldiagnostic information or to intervene in disease processes. In aspecific embodiment, individuals at risk for cancer or to developautoimmune diseases will display a PGS-2 related resistance to celldeath upon stimulation of cells by chemical factors including, but notlimited to, TNFα and FAS ligand. By intervening in this process by, forexample, the administration of inhibitors of PGS-2-related activity, itis possible to facilitate the completion of cell death events toeliminate inappropriate cells from the biological milieu. In this way,autoimmune T-cells can be removed through the apoptic mechanism uponstimulation by appropriate chemical signals or immunization withdisease-related target antigens such as insulin or GAD. Similarly,cancer cells can also proceed to appropriate cell death, therebypreventing or reducing tumors and/or other inappropriate cellularproliferation.

Thus, a further specific embodiment of the subject invention concernsthe administration of inhibitors of PGS-2, or its biological activities,to effect a modulation of programmed cell death such thatself-destructive T-cells and/or cancer cells are removed to reduce orprevent autoimmune or cancer conditions. The inhibitors of PGS-2 whichcan be used according to the subject invention include, but are notlimited to, glucocorticoid hormones (which suppress expression ofPGS-2), IL-10, IL4, IL-13, and TGF-β.

In a further embodiment of the subject invention modulation of celldeath can be achieved by upregulating the cellular response resultingfrom activation of the FAS receptor. This upregulation can be achievedby administration of an agent which increases FAS receptor expression.This agent may be, for example, a PGE inhibitor. Alternatively, theupregulation of the FAS cellular response can be achieved by, forexample, administration of agents which enhance the intracellularresponse to FAS receptor activation. Thus, in individuals having apathological condition attributable to aberrant cell death processes,cell death can be promoted by upregulating the intracellular cascade ofevents whereby FAS receptor activation ultimately promotes cell death.This upregulation can be achieved by those skilled in the art, havingbenefit of the instant disclosure, by, for example, stimulatingenzymatic and other regulatory molecules which participate in the FASactivation pathway. In a specific embodiment, individuals identified asneeding increased T-cell death can be treated to increase both CD25expression and cellular response to FAS receptor activation. Thistherapy can be further augmented by administration of an appropriateantigen thereby enhancing CD25 activation and increasing the specificityof the treatment. The antigen may be, for example, an autoantigen.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the blocking of PGE₂ production by the PGS-2-specificinhibitor, NS-398.

FIG. 2 shows the correlation between increased PGS-2 levels and anincreased risk of IDD.

FIG. 3 shows the ability of a PGS-2 inhibitor to increase expression ofCD25.

DETAILED DISCLOSURE OF THE INVENTION

In one aspect the subject invention pertains to the detection and/ormodulation of prostaglandin synthase-2 (PGS-2) production inantigen-presenting cells (APCs) of individuals at risk for developingautoimmune disease or cancer. In this embodiment, the subject inventionconcerns the discovery that PGS-2 is a cellular marker for individualsat risk for IDD and other autoimmune diseases. Another embodiment of thesubject invention concerns preventative or therapeutic treatments. In apreferred embodiment, drugs that block PGS-2 production or activity canbe administered to individuals who have autoimmune disease or cellularproliferation disease, or are at risk for developing such diseases. In afurther embodiment, the subject invention pertains to monitoring and/ormodulation of CD25 expression and/or FAS receptor activation of T-cellsor other cells having dysfunctional cell death processes.

The subject invention provides quick and easy procedures for determiningwhether an individual may be at risk for developing autoimmune disease.In one diagnostic aspect of the invention, peripheral blood monocytescan be evaluated to determine the level of PGS-2 expression. Thisevaluation can be conducted using any one of a number of diagnosticprocedures well known to those skilled in the art. These procedures maybe used to detect PGS-2 directly or to detect evidence of PGS-2expression. Evidence of PGS-2 expression includes, for example, thepresence of PGS-2 mRNA. The PGS-2 mRNA can be detected by, for example,reverse transcriptase PCR (RT-PCR). PGE₂, which is produced on accountof the enzymatic activity of PGS-2, can readily be detected using, forexample, ELISA, RIA, or other antibody-based assays. The expression ofPGS-2 protein can also be detected using flow cytometry methods orWestern blotting.

In a further diagnostic aspect of the subject invention, T-cells areevaluated to determine levels of CD25 expression and/or FAS receptorexpression. A decrease in expression of either CD25 or FAS receptor hasbeen found to be indicative of risk for autoimmune disease—particularlyIDD. A decrease in the combined expression of CD25 and FAS receptor isparticularly indicative of risk for autoimmune disease. The level ofexpression of these molecules can be readily ascertained by thoseskilled in the art using standard techniques such as, for example, FACSanalysis using commercially available antibodies. A further diagnosticprocedure involves the determination of the increase in CD25 expressionresulting from the administration of a PGE-2 inhibitor. An increase inCD25 expression on T-cells upon treatment with a PGE-2 inhibitor isindicative of risk for autoimmune disease including IDD. The PGE-2inhibitor may be, for example, NS398 or indomethacin, which inhibitsboth PGS-1 and PGS-2.

The diagnostic assays of the subject invention can be used to detectevidence of autoimmune disease before the appearance of clinicalsystems. Furthermore, the assays are useful for monitoring diseaseprogression or the effect of treatment.

In a therapeutic and/or preventative aspect of the subject invention,the action of prostaglandins can be blocked or inhibited, therebyslowing or eliminating undesirable immune processes. In a preferredembodiment, the activity of PGS-2 and/or related molecules can beblocked. Indomethacin is known to block the activity of PGS-2.Aminoguanidine is an inhibitor of inducible nitric oxide synthase(iNOS). NO augments PGS-2 activity. Thus, the subject invention providesa method for reducing the severity of autoimmune disease. As used hereinreference to “reducing the severity” of a disorder would includepreventing or delaying the disorder or making the effects of thedisorder less damaging physically or emotionally.

Treatment of NOD mice afflicted with active, established autoimmunedisease using a drug combination of indomethacin and aminoguanidinemarkedly delays the onset and reduces the incidence of diabetes. Thecombination of inhibitors is particularly effective. Furthermore,treatment of NOD mice at an early stage of disease with indomethacinalone effectively blocks the development of IDD. It has also been foundthat the PGS-2-specific inhibitor, NS-398 (available from CaymanChemical Company), effectively blocks all PGS-2 production in vitro frommonocytes of individuals at high risk for IDD. Treatment of individualsat high risk for IDD can be used to block the progression ofautoimmunity to frank diabetes.

The treatments of the subject invention are also useful to reduce theseverity of the side effects of autoimmune disease. In a particularlypreferred embodiment the side effects of diabetes can be reduced bytreatment with a prostaglandin inhibitor.

In a further embodiment of the subject invention, PGS-2-specificinhibitors can be used in conjunction with antigen-specific immunizationtherapy. In the immunization treatment paradigms, a target antigen forIDD, or other autoimmune disease, is used to immunize the individual.This results in tolerance and a lack of progression to overt disease. Inthe case of diabetes, antigens useful in this regard include, but arenot limited to, insulin, GAD, IA-2, IA-2β, and fragments and variants ofthese antigens. Antigens associated with various autoimmune conditionsinclude, but are not limited to, those shown below in Table 1.

TABLE 1 Condition Antigens Multiple Sclerosis myelin basic proteinproteolipid protein Rheumatoid Arthritis collagen Lupus DNA histoneproteins IDDM GAD insulin IA-2 IA-2β 38 kD protein perforin Thyroidthyroglobulin peroxidase Vitiligo tyrosinase

As PGs inhibit lymphocyte activation, and activation is a prerequisitefor apoptosis of T cells, treatment of subjects with PGS-2 inhibitorsprior to, or concurrent with, immunization can potentiate the effects ofthis therapy. Therefore, PGS-2 inhibitors can be used as an adjuvanttherapy for antigen immunization to prevent IDD or other autoimmunediseases.

The monocyte production of the monokines IL-1β, TNFα, IL-1RA, and IL-10in culture supernatants from healthy controls and the pre-diabeticpopulation in the presence and absence of the PGS-2-specific inhibitor,NS-398, have been evaluated. It has been determined that NS-398 promotesthe production of the immunoregulatory monokines IL-10 and IL-1RA,suggesting that high levels of PGE₂ produced by pre-diabetic monocytesresult in PGE₂ desensitization. It has also been determined that,despite enhanced levels of PGS-2 and PGE₂ in diabetic and pre-diabeticindividuals, there is no corresponding increase in IL-10. Furthermore,in NOD mice, administration of IL-10 surprisingly does not inhibit PGE₂production by monocytes. Therefore, a further aspect of the subjectinvention is a method for detecting evidence of autoimmune disease,particularly diabetes, which comprises evaluating monocytes to determineif those monocytes have a reduced response to IL-10 with regard to PGS-2expression. Such reduced response would be evidence of disease. Inaccordance with these findings, compounds that inhibit prostaglandinproduction can be used to modulate the production of anti-inflammatorymonokines in order to limit the immune response. The spontaneousexpression of PGS-2 and the production of PGE₂ by pre-diabetic monocytesmay influence their function, limiting their ability to producemonokines that would have a potent effect on limiting the autoimmuneresponse to the β-cells of the islet and therefore would promote theprogression to diabetes.

Treatments that inhibit PGE₂ can be used according to the subjectinvention to restore the production of these potent regulatory monokinesand either slow or block the autoimmune process, as well as to promoteappropriate apoptotic processes. In addition, the production of IL-10 bythe monocyte can be used to promote the production of Th2 lymphocytesthat are thought to play an important regulatory role in IDD.Furthermore, blocking PGE₂ and limiting prostaglandin desensitizationcan also be used to promote the generation of TH2 cells as PGE₂ promotesthe generation of these cells.

A further therapeutic embodiment of the subject invention pertains tothe modulation of CD25 expression of T-cells. In a related aspect of thesubject invention the cellular response to FAS receptor activation canbe modulated. For individuals at risk for IDD or in need of increasedprogrammed cell death, the subject invention provides a treatmentwhereby the efficiency of the cell death pathway is enhanced. Thisenhancement is accomplished by increasing expression of CD25 and/or theresponse to FAS receptor activation. In a preferred embodiment, theenhancement of CD25 expression can be accomplished by administering aninhibitor of PGE₂. Upregulation of FAS-related cellular activation ispreferably achieved by increasing expression of the FAS protein or byenhancing the amount, or activity, of compounds which promote theintracellular FAS activation pathway. In one embodiment, this therapy isaugmented by the administration of an antigen thereby enhancing theactivity resulting from expression of CD25. The antigen may be, forexample, an autoantigen.

In a specific embodiment, the subject invention comprises administeringa prostaglandin inhibitor to an individual who has been determined to beat risk for developing autoinmmune disease. In a preferred embodiment,the prostaglandin inhibitor is administered chronically. In a furtherpreferred embodiment, the prostaglandin inhibitor is administered at adose which is higher than that which would be used, for example, torelieve pain or inflammation. In this regard, the inhibitor should beadministered at a dosage and in a manner which will effectively increaseexpression of CD25 on T-cells. In a particularly preferred embodiment,the prostaglandin inhibitor is an inhibitor of PGS-2. The therapy of thesubject invention is particularly advantageous when administered priorto the appearance of clinical symptoms of autoimmune disease or the needfor enhanced programmed cell death.

Materials and Methods

RT-PCR detection of PGS-2 mRNA in human monocytes. Human peripheralblood is collected in sterile green top 10 ml tubes. Assays arepreferably performed with 5 ml or more of blood. The blood is thencentrifuged on a ficoll gradient for 30 minutes at 1500 rpm. Monocytesare isolated to >80% purity by adherence for 2 hours to a plasticculture surface. Cells are then cultured overnight in RMPI-1640 plusendotoxin-free fetal calf sera. The cells are harvested after 16 hoursof culture by cold Ca⁺⁺/Mg⁺⁺-free PBS, the cells are counted, andviability is assessed. Monocytes cultured in 10 μg/ml LPS serve as apositive control for each sample tested. The poly MRNA is then harvestedfrom a standard 10⁵ monocytes using a kit (Quiagen). The mRNA is thenreverse transcribed in a standard reaction mixture. The cDNA is thenamplified by PGS-2-specific primers designed by our laboratory using astandard reverse transcriptase reaction for 45 cycles. The PCR productis then run out on agarose gels with known standards and the sizeconfirmed. β-actin is used as an internal control in these reactions.The identity of RT-PCR products were confirmed by size and Southernblotting using a PGS-2-specific labeled internal probe.

PGS-2 protein detection in human monocytes. A PGS-2-specific mousemonoclonal antibody (Cayman Chemical Company) has been utilized fordetection of PGS-2 protein in monocytes. Cells for immunocytochemistryare adhered to multichamber slides for two hours, fixed in 0.2%paraformaldehyde, and permeabilized with Triton-X and glycine in PBS for10 minutes. The primary antibody is incubated overnight at 4° C. andthen detected with FITC labeled Fab goat anti-rabbit antisera. The cellsare then visualized with a fluorescent microscope.

A fluorescent activated cell sorting (FACs) method can also be used fordetection of PGS-2 in monocytes. In this procedure, whole blood islabeled with an anti-monocyte antibody CD14 coupled to a phycoerythrinmolecule. The cells are then fixed and lysed with FACs-Lyse(Becton-Dickenson). The cells are then further permeabilized withsaponins throughout the procedure, and then analyzed on a FACs machine.The percent of monocytes positive for PGS-2, as well as fluorescentintensity, is then determined. Approximately 15%-70% of peripheral bloodmonocytes from ICA+ individuals are positive by this method.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Detection of PGS-2 mRNA

The regulation of mRNA and protein expression of PGS-2, the inducibleenzyme critical for the production of large quantities ofprostaglandins, was examined in macrophages of the NOD mouse.Macrophages from control mouse strains did not express PGS-2 mRNA asdetermined by the highly sensitive reverse transcriptase polymerasechain reaction. The PGS-2 protein is likewise not expressed in restingcontrol macrophages as assessed by indirect immunofluorescence using aPGS-2 specific antibody. In marked contrast, NOD macrophagesspontaneously express high levels of mRNA and protein for this enzyme asdetermined by these techniques. PGS-2 was also found to be expressed inthe macrophages of NODscid/scid mice which lack functional T and B cellsand, as a result, do not develop autoimmune disease or diabetes. Theexpression of PGS-2 in macrophages of NODscid/scid mice indicates thatspontaneous PGS-2 expression is not dependent on the autoimmune milieu,and suggests that PGS-2 expression is a result of a primary macrophagedefect. Therefore, the aberrant expression of PGS-2 by NOD macrophagesreadily explains the abnormal PG production by these cells and providesthe molecular and cellular basis for APC dysfunction.

EXAMPLE 2 Role of PGS-2 Expression in IDD

In order to more fully establish the genetic contribution of PGS-2expression to autoimmunity in the NOD mouse, congenic mice wereexamined. The gene encoding PGS-2 is located on chromosome 1, 76.2 CMfrom the centromere. These mice, designated B6.NOD.C1, have a segment ofchromosome 1 from the NOD mouse that contains the “NOD PGS-2 gene,”while the rest of the mouse genome is from the non-autoimmune C57BL/6mouse. Macrophages from B6.BOD.C1 congenic mice, like the NOD,spontaneously express PGS-2 mRNA and protein. These mice developautoimmune disease in the pancreas, but unlike the NOD, do not developdiabetes. The absence of diabetes in these congenic mice is likely dueto a lack of other key genes from the NOD that contribute otherimportant factors to the overall disease process. Another line ofcongenic mice were designated NOD.B10.C1. These mice contain the NODgenome except for a segment of chromosome 1 derived from thenon-autoimmune C57BL/10 mouse and contains the “C57BL/10 PGS-2 gene.”NOD.B10.C1 mice do not spontaneously express PGS-2 and have a 40-50%reduction in the incidence of diabetes. These data, along with thosefrom the B6.NOD.C1 congenic mice suggest that the expression of PGS-2correlates with a more aggressive autoimmune phenotype.

EXAMPLE 3 Blocking Activity of PGS-2 as Therapy for IDD

Blocking the activity of PGS-2 with drugs that reduce PG production canbe used to prevent or slow the development of IDD disease. When NOD micewith established, active autoimmune disease are treated through theirdrinking water with a combination of drugs, including a PGS-1/PGS-2inhibitor, low dose indomethacin (3 μg/ml), in conjunction with aninducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine, whichpotentiates the effects of indomethacin, the incidence of diabetes inNOD mice drops by 42% in comparison to control animals and animalstreated with either low dose indomethacin or aminoguanidine alone.

Treatment of NOD mice at a time when autoimmunity is in its final stageswith high doses (15 μg/ml) of indomethacin alone reduces the incidenceof diabetes from 77% to 22%. These data demonstrate a strong effect ofPGS inhibitors on the development of IDD.

EXAMPLE 4 Association of PGS-2 and PGE₂ with Autoimmune Disease

The effects of non-steroidal anti-inflammatory drugs (NSAIDs) thatspecifically inhibit PGS-2 in vitro were assessed, and they were foundto be highly potent blockers of NOD mouse macrophage PGS-2 production.Aspirin-like drugs that inhibit both PGS-1 and PGS-2 cause gastricirritation, whereas PGS-2-specific drugs do not.

Monocytes were examined from ICA+ humans with a high risk of developingIDD, individuals with established SLE and autoimmune thyroid disease,and healthy controls. A portion of the pre-diabetic ICA+ individualsexamined are enrolled in an IDD trial where they receive dailysubcutaneous insulin as a preventative therapy. Monocytes of humanswithout autoimmune disease infrequently express PGS-2 mRNA (12%),whereas monocytes from subjects who are at high risk for the developmentof IDD express PGS-2 at a highly significant frequency (84%, p<0.0001).The preventative insulin therapy does not appear to affect PGS-2 mRNA orprotein expression (see Table 2).

TABLE 2 PGS-2 mRNA expression in MOs of healthy controls and ICA⁺subjects N PGS-2 + % Positive Healthy controls 25 3 12% Females 12 0  0%Males 13 3 23% ICA⁺ 31 26  84%* Females 17 12 80% Males 14 11 85%Insulin RX 14 10 71% No RX 17 16 94% Significant difference (p<0.0001)analyzed by a two-tailed Fisher Test.

PGS-2 expression, however, is not specific for IDD, as humans with SLEand autoimmune thyroid disease also spontaneously express PGS-2 in theirmonocytes (see Table 3)

TABLE 3 PGS-2 expression in MOs of autoimmune controls N PGS-2 + %Positive SLE 5 4 80% Hashimoto's 4 2 50%

In addition to the expression of PGS-2, the production of PGE2 bymonocytes from these same control and autoimmune individuals wasexamined. It was found that PGE2 production, as determined by specificELISA, is significantly higher in ICA+ individuals and in autoimmunecontrols than in control monocytes (p<0.0001) (see Table 4).

TABLE 4 PGE₂ production by MOs of healthy controls, ICA⁺, and SLEsubjects N PGE₂ pg/ml Healthy controls 18  789 +/− 243* ICA⁺/PGS-2 (+)23  7705 +/− 1510* ICA⁺/PGS-2 (−) 5 288 +/− 211 ICA⁺/PGS-2 +/Ins. RX 105805 +/− 1544 SLE/PGS-2 + 4 15437 +/− 12900 *Significant difference(p<0.0001) as analyzed by two-tailed Fisher Test.

Finally, the production of PGE2 was completely blocked by thePGS-2-specific inhibitor NS-398 (see FIG. 1).

EXAMPLE 5 Monocyte Production of TNFα and IL-1β

The expression of PGS-2 can be induced by several factors includingcytokines produced by monocytes such as TNFα and IL-1β. To evaluate thepossibility that monocytes of pre-IDD subjects produce large quantitiesof IL-1 and TNFα which induces PGS-2, the levels of these cytokines weremeasured in supernatants of monocytes cultured in vitro for 24 hoursfrom pre-IDD and healthy controls. As summarized in the table below, itwas found that the levels of both IL-1 and TNFα produced by monocyteswere actually lower in the pre-IDD subjects than in the controls (seeTable 5). The lower levels of these cytokines is consistent with theconstitutive production of prostaglandins which suppress the productionof both TNFα and IL-1β. A comparison of TNFα and IL-1β levels betweeninsulin-treated and untreated pre-IDD yielded no significant differencesbetween these two groups.

TABLE 5 N pg/ml IL-1β: Healthy controls 13 1455 ± 585 ICA + pre-IDD 131189 ± 325 TNFα: Healthy controls 10  718 ± 345 ICA + pre-IDD 14 282 ±91

IL-10 production by monocytes of pre-IDD and healthy controls were alsoexamined. It was found that there was no significant difference in thelevels of IL-10 produced by either resting or stimulated monocytes frompre-IDD or healthy controls.

These data demonstrate that PGS-2 expression is not secondary to highlevels of IL-1β or TNFα production or the lack of IL-10 secretion bypre-IDD monocytes. These findings support the notion that PGS-2expression in the pre-IDD monocyte is a primary defect.

EXAMPLE 6 PGS-2 mRNA Expression

PGS-2 mRNA expression has been found to be stable in the pre-IDDsubjects as five individuals examined on more than one occasion (usually3-6 months from the previous observation) remain positive. Likewise, sixcontrol subjects negative for PGS-2 remain negative with similarfollow-up testing (see Table 6).

TABLE 6 Subject group PGS-2+/Total % PGS-2+ Healthy controls  2/23 8.6ICA + pre-IDD (observation) 14/16 87.5* ICA + pre-IDD (insulin-treated)15/19 73.6** ICA negative insulin autoantibody positive 2/5 40 ICAnegative established IDD (>5 years) 6/7 85.7*** Systemic lupuserythematosus 5/7 71**** Autoimmune thyroiditis 2/3 66 *vs. controls p =0.0024 (unpaired Fisher's T test) **vs. controls p = 0.0032 ***vs.controls p = 0.025 ****vs. controls p = 0.011

EXAMPLE 7 PGS-2 Expression as Primary or Secondary Defect

Six long-term IDD patients (diabetes onset>5 years) and found that ⅚ ofthese individuals express PGS-2 have been analyzed. Studies show thatICA is lost in IDD patients within five years of the onset of clinicaldiabetes, reflecting a “burned out” autoimmune process. These data lendfurther support to the notion that PGS-2 is a primary monocyte defect inhuman IDD as it is in the NOD mouse.

Identification of PGS-2 as a primary defect enables the use of PGS-2 asan early cellular marker for IDD susceptibility. Differences in thePGS-2 gene of normal individuals and autoimmune subjects can be used toperform genetic screening of individuals to assess susceptibility todiabetes or other autoimmune disease.

Regardless of its status as a primary defect, PGS-2 expression reflectsan active autoimmune process, and is highly advantageous in identifyingindividuals at high risk for IDD. In this regard, PGS-2-positiveindividuals who produce the highest levels of PGS-2 have progressed toclinical diabetes the fastest. It is also known that autoimmune diseasesprogress into spontaneous remissions or exacerbations. PGS-2 expressioncan be used to identify and/or monitor such changes in disease activity,i.e., PGS-2 positively reflecting higher levels of disease activity andloss of PGS-2 expression reflecting remission. This is of greatimportance in IDD, where no physical signs or symptoms manifest tosuggest exacerbation of the autoimmune attack on the insulin-producingcells.

PGS-2 is expressed in a high percentage of monocytes from individualswith autoimmune disorders such as SLE and thyroiditis. Thus, PGS-2expression in monocytes can be employed as a cellular marker for otherautoimmune diseases in addition to IDD. Of note in screening healthycontrols is one individual whose monocytes were strongly positive forPGS-2 expression, who had no personal or family history of autoimmunediseases. This individual, six weeks post-screening, developed Raynaud'sphenomenon and a strongly positive ANA, suggesting the development of acollagen vascular disease. This further supports the utility of thesubject invention for general screening for autoimmune dysfunction.

EXAMPLE 8 FACS Assay for PGS-2

One aspect of the invention is a fluorescent activated cell sorter(FACS) based assay for PGS-2 protein. Using this assay, it is possibleto quantitate the percentage of cells in the peripheral blood thatexpress the PGS-2 protein. This assay employs an antibody thatspecifically binds to the PGS-2 protein. The binding of this antibody toPGS-2 can be detected because the antibody is coupled to a fluorescentmolecule which can be detected by the lasers of the FACS machine. Usingthis procedure, which requires only one-half teaspoon of blood, it ispossible to detect the expression of PGS-2 protein in blood cells anddetermine the percentage of monocytes of pre-IDD individuals thatconstitutively express this enzyme.

EXAMPLE 9 Uses, Formulations, and Administrations

Application of the treatments of the subject invention can beaccomplished by any suitable method and technique presently orprospectively known to those skilled in the art.

In one embodiment, compounds of the subject invention have effectiveimmunomodulatory activity. Specifically, they are useful in regulatingimmune responses in animals and humans. Thus, pharmaceuticalcompositions containing compounds of the invention as active ingredientsare useful in prophylactic or therapeutic treatment of animmunomodulatory response in humans or other mammals.

The dosage administered will be dependent upon the immunomodulatoryresponse desired; the type of host involved; its age, health, weight,kind of concurrent treatment, if any; frequency of treatment;therapeutic ration and like considerations.

The compounds of the subject invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.In general, the compositions of the subject invention will be formulatedsuch that an effective amount of the bioactive compound(s) is combinedwith a suitable carrier in order to facilitate effective administrationof the composition.

In accordance with the invention, pharmaceutical compositions comprisingan active ingredient and one or more non-toxic, pharmaceuticallyacceptable carrier or diluent.

The compositions of the invention are advantageously used in a varietyof forms, e.g., tablets, capsules, pills, powders, aerosols, granules,and oral solutions or suspensions and the like containing suitablequantities of the active ingredient. Such compositions are referred toherein and in the accompanying claims generically as “pharmaceuticalcompositions.” Typically, they can be in unit dosage form, namely, inphysically discrete units suitable as unitary dosages for human oranimal subjects, each unit containing a predetermined quantity of activeingredient calculated to produce the desired therapeutic or prophylacticeffect in association with one or more pharmaceutically acceptable otheringredients, e.g., diluent or carrier.

It should be understood that the example and embodiments describedherein are for illustrative purpose only and that various modificationsor changes in light thereof will be suggested to persons skill in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

What is claimed is:
 1. A method for detecting susceptibility toautoimmune disease in a human or animal, or monitoring the progressionof such disease, wherein said method comprises assayingantigen-presenting cells for enhanced expression of prostaglandinsynthase-2 (PGS-2).
 2. The method, according to claim 1, wherein saidexpression of PGS-2 is evidenced by the presence of PGS-2 messenger RNA.3. The method, according to claim 2, which comprises detection of saidPGS-2 messenger RNA by reverse transcriptase PCR.
 4. The method,according to claim 1, wherein said antigen presenting cells are selectedfrom the group consisting of macrophages and monocytes.
 5. The method,according to claim 4, wherein said monocytes are peripheral bloodmonocytes.
 6. The method, according to claim 1, wherein said assayingfor enhanced expression of PGS-2 comprises assaying saidantigen-presenting cells for the presence of enhanced levels of PGE₂.